De Novo Transcriptome Sequencing of Codonopsis lanceolata for Identification of Triterpene Synthase and Triterpene Acetyltransferase.

Codonopsis lanceolata (Campanulaceae) is a perennial plant commonly known as the bonnet bellflower. This species is widely used in traditional medicine and is considered to have multiple medicinal properties. In this study, we found that shoots and roots of C. lanceolata contained various types of free triterpenes (taraxerol, ß-amyrin, α-amyrin, and friedelin) and triterpene acetates (taraxerol acetate, ß-amyrin acetate, and α-amyrin acetate). The content of triterpenes and triterpene acetates by GC analysis was higher in the shoot than in the roots. To investigate the transcriptional activity of genes involved in triterpenes and triterpene acetate biosynthesis, we performed de novo transcriptome analysis of shoots and roots of C. lanceolata by sequencing using the Illumina platform. A total of 39,523 representative transcripts were obtained. After functional annotation of the transcripts, the differential expression of genes involved in triterpene biosynthetic pathways was investigated. Generally, the transcriptional activity of unigenes in the upstream region (MVA and MEP pathway) of triterpene biosynthetic pathways was higher in shoots than in roots. Various triterpene synthases (2,3-oxidosqualene cyclase, OSC) participate to produce triterpene skeletons by the cyclization of 2,3-oxidosqualene. A total of fifteen contigs were obtained in annotated OSCs in the representative transcripts. Functional characterization of four OSC sequences by heterologous expression in yeast revealed that ClOSC1 was determined as taraxerol synthase, and ClOSC2 was a mixed-amyrin synthase producing α-amyrin and ß-amyrin. Five putative contigs of triterpene acetyltransferases showed high homology to the lettuce triterpene acetyltransferases. Conclusively, this study provides the basis of molecular information, particularly for the biosynthesis of triterpenes and triterpene acetates in C. lanceolata.

Enhanced monoterpene emission in transgenic orange mint (Mentha × piperita f. citrata) overexpressing a tobacco lipid transfer protein (NtLTP1).

MAIN CONCLUSION: Overexpression of the tobacco lipid transfer protein (NtLTP1) gene in transgenic orange mint resulted in enhanced accumulation of monoterpenes in the cavity of head cells of glandular trichomes, which resulted in enhanced emission of monoterpenes from transgenic orange mints. Plants in the genus Mentha (Lamiaceae) produce volatile oils that accumulate in peltate glandular trichomes in the aerial parts of plants. A lipid transfer protein (NtLTP1) in tobacco showed glandular trichome-specific expression and supported the secretion of diterpenoid lipids from head cells of glandular trichomes (Choi et al., Plant J 70:480-491,2012). Here, we constructed transgenic orange mint (Mentha × piperita f. citrata) overexpressing the tobacco NtLTP1 gene via Agrobacterium-mediated transformation. Transgenic lines of orange mint overexpressing NtLTP1 were confirmed by genomic PCR and RT-PCR. Immunoblotting analysis using an NtLTP1 polyclonal antibody showed clear dark spots at the position of the lipid exudates from tobacco glandular trichomes and the squeezed out lipids from the glandular trichomes of transgenic orange mint. Heads of glandular trichomes in transgenic plants overexpressing the NtLTP1 gene showed a larger diameter than those of the wild-type control. The enhanced size of trichome heads in transgenic orange mint was confirmed by scanning electron microscopy. Volatile components were extracted from wild-type and transgenic orange mint by solid-phase microextraction (SPME) and analyzed by headspace-gas chromatography-mass spectrometry (HS/GC/MS). Linalyl acetate was the most abundant component among the eleven identified monoterpenes in the volatile compounds extracted from both the wild-type and transgenic lines of orange mint. Overexpression of NtLTP1 in transgenic orange mint plants resulted in enhanced emission of volatile monoterpenoids compared with that of volatile monoterpenoids in the wild-type control plants.

Lipid Transfer Proteins (AaLTP3 and AaLTP4) Are Involved in Sesquiterpene Lactone Secretion from Glandular Trichomes in Artemisia annua.

In Artemisia annua plants, glandular trichomes (GTs) are responsible for the biosynthesis and secretion of sesquiterpene lactones including artemisinin/arteannuin B. Nonspecific lipid transfer proteins (LTPs) in plants bind and carry lipid molecules across the cell membrane and are also known as secretary proteins. Interestingly, the transcripts of LTP genes are exceptionally abundant in the GTs of A. annua. In the present study, we isolated two trichome-specific LTP genes (AaLTP3 and AaLTP4) from a Korean ecotype of A. annua. AaLTP3 was expressed abundantly in shoots, whereas AaLTP4 was expressed in flowers. The GUS signal driven by the AaLTP3 or AaLTP4 promoter in transgenic A. annua plants revealed that the AaLTP3 promoter was active on hair-like non-GTs and that the AaLTP4 promoter was active on GTs. Analysis of enhanced cyan fluorescent protein (ECFP) fluorescence fused with the AaLTP3 or AaLTP4 protein in transgenic tobacco revealed that ECFP florescence was very bright on secreted lipids of long GTs. Moreover, the florescence was also bright on the head cells of short trichomes and their secreted granules. Immunoblotting analysis of GT exudates in petioles of A. annua revealed a strong positive signal against the AaLTP4 antibody. Overexpression of AaLTP3 or AaLTP4 in transgenic A. annua plants resulted in enhanced production of sesquiterpene lactones (arteannuin B, artemisinin, dihydroartemisinic acid and artemisinic acid) compared with those of wild type. The present study shows that LTP genes (AaLTP3 or AaLTP4) play important roles in the sequestration and secretion of lipids in GTs of A. annua, which is useful for the enhanced production of sesquiterpene lactones by genetic engineering.

Cloning and Characterization of Oxidosqualene Cyclases Involved in Taraxasterol, Taraxerol and Bauerenol Triterpene Biosynthesis in Taraxacum coreanum.

Triterpenes, consisting of six isoprene units, are one of the largest classes of natural compounds in plants. The genus Taraxacum is in the family Asteraceae and is widely distributed in the Northern Hemisphere. Various triterpenes, especially taraxerol and taraxasterol, are present in Taraxacum plants. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene after the rearrangement of the triterpene skeleton. However, no functional characterization of the OSC genes involved in triterpene biosynthesis, except for a lupeol synthase in Taraxacum officinale, has been performed. Taraxacum coreanum, or Korean dandelion, grows in Korea and China. Putative OSC genes in T. coreanum plants were isolated by transcriptome analysis, and four of these (TcOSC1, TcOSC2, TcOSC3 and TcOSC4) were functionally characterized by heterologous expression in yeast. Both TcOSC1 and TcOSC2 were closely related to dammarenediol-II synthases. TcOSC3 and TcOSC4 were strongly grouped with ß-amyrin synthases. Functional analysis revealed that TcOSC1 produced several triterpenes, including taraxasterol; Ψ-taraxasterol; α-, ß- and δ-amyrin; and dammarenediol-II. TcOSC2 catalyzed the production of bauerenol and another unknown triterpene, TcOSC3 catalyzed the production of ß-amyrin. TcOSC4 catalyzed the production of taraxerol. Moreover, we identified taraxasterol, ψ-taraxasterol, taraxerol, lupeol, δ-amyrin, α-amyrin, ß-amyrin and bauerenol in the roots and leaves of T. coreanum. Our results suggest that TcOSC1, TcOSC2, TcOSC3 and TcOSC4 are key triterpene biosynthetic enzymes in T. coreanum. These enzymes are novel triterpene synthases involved in the production of taraxasterol, bauerenol and taraxerol.

Genetically modified rice produces ginsenoside aglycone (protopanaxadiol).

MAIN CONCLUSION: Protopanaxadiol is dammarane-type tetracyclic triterpene sapogenin found in ginseng and has a high medicinal values. We successfully constructed transgenic rice producing protopanaxadiol by introducing the ginseng PgDDS and CYP716A47 genes in this crop plant. Protopanaxadiol (PPD), an aglycone of ginsenosides, possesses pleiotropic anticarcinogenesis activities in many cancers. Here, we constructed transgenic rice overexpressing the Panax ginseng dammarenediol-II synthase gene (PgDDS) and protopanaxadiol synthase gene (CYP716A47) driven by a rice endosperm-specific α-globulin promoter. Among more than 50 independent lines, five transgenic lines were selected. The introduction of the genes in the T1 generation of the transgenic lines was confirmed by genomic PCR. The expression of the introduced genes in T2 seeds was confirmed by qPCR. Methanol extracts of transgenic rice grains were analyzed by LC/MS to detect the production of PPD and dammarenediol-II (DD). The production of both PPD and DD was identified not only by comparing the retention times but also mass fraction patterns of authentic PPD and DD standards. The mean concentrations of PPD and DD in rice grains were 16.4 and 4.5 µg/g dry weight, respectively. The invention of genetically engineered rice grains producing PPD and DD can be applied to rice breeding to reinforce new medicinal values.

Functional characterization of an oxidosqualene cyclase (PdFRS) encoding a monofunctional friedelin synthase in Populus davidiana.

MAIN CONCLUSION: An oxidosqualene cyclase (PdFRS) from Populus davidiana was characterized as a monofunctional friedelin synthase by its heterologous expression in yeast and overexpression in plants. Triterpenes are one of the largest classes of plant chemical compounds composed of three terpene units, which form the basic skeleton of all sterols and saponins. Friedelin (friedelan-3-one), a pentacyclic triterpene, occurs in many plant families and is particularly present in rich amounts in cork tissues from trees. The biosynthesis of friedelin occurs through the oxidosqualene cyclase (OSC) enzyme that generates friedelin from 2,3-oxidosqualene after the maximum rearrangement of a triterpene skeleton. Populus davidiana is called Korean aspen and grows in northern East Asia. From 57,322 unique sequences generated from the P. davidiana transcriptome database, one complete coding sequence (PdFRS) was obtained from a contig, which showed 74% identity to Betula platyphylla ß-amyrin synthase and 73% identity with friedelin synthase from Maytenus ilicifolia. The open reading frame (ORF) region of the PdFRS sequence was 2280 bp long and composed a 759 amino acid protein with a predicted molecular mass of 87.81 kDa. qPCR analysis revealed that methyl jasmonate treatments strongly upregulated PdFRS gene expression and resulted in enhanced friedelin accumulation in leaves. Heterologous expression of the PdFRS gene in yeast resulted in the production of friedelin triterpene as a single product, which was confirmed by comparison with the mass fragmentation pattern from an authentic friedelin standard by GC/MS analysis. Transgenic P. davidiana overexpressing the PdFRS gene was constructed via Agrobacterium-mediated transformation. Overexpression of PdFRS in transgenic P. davidiana lines resulted in enhanced friedelin production.

Transcriptomic Analysis of Kalopanax septemlobus and Characterization of KsBAS, CYP716A94 and CYP72A397 Genes Involved in Hederagenin Saponin Biosynthesis.

Kalopanax septemlobus, commonly named the castor aralia tree, is a highly valued woody medicinal tree belonging to the family Araliaceae. Kalopanax septemlobus contains approximately 15 triterpenoid saponins primarily constituted of hederagenin aglycones. Hederagenin is a representative precursor for hemolytic saponin in plants. In the present study, transcriptome analysis was performed to discover genes involved in hederagenin saponin biosynthesis in K. septemlobus. De novo assembly generated 82,698 unique sequences, including 17,747 contigs and 64,951 singletons, following 454 pyrosequencing. Oxidosqualene cyclases (OSCs) are enzymes that catalyze the formation of diverse triterpene skeletons from 2,3-oxidosqualene. Heterologous expression of an OSC sequence in yeast revealed that KsBAS is a ß-amyrin synthase gene. Cytochrome P450 genes (CYPs) make up a supergene family in the plant genome and play a key role in the biosynthesis of sapogenin aglycones. In total, 95 contigs and 110 singletons annotated as CYPs were obtained by sequencing the K. septemlobus transcriptome. By heterologous expression in yeast, we found that CYP716A94 was ß-amyrin 28-oxidase involved in oleanolic acid production from ß-amyrin, and CYP72A397 was oleanolic acid 23-hydroxylase involved in hederagenin production from oleanolic acid. Engineered yeast co-expressing KsBAS, CYP716A94 and CYP72A397 produced hederagenin. Kalopanax septemlobus CYP72A397 is a novel CYP enzyme that synthesizes hederagenin aglycone from oleanolic acid as a single product. In conclusion, we characterized three genes participating in sequential steps for hederagenin biosynthesis from ß-amyrin, which are likely to play a major role in hederagenin saponin biosynthesis in K. septemlobus.

Heterologous production of a ginsenoside saponin (compound K) and its precursors in transgenic tobacco impairs the vegetative and reproductive growth.

MAIN CONCLUSION: Production of compound K (a ginsenoside saponin) and its precursors in transgenic tobacco resulted in stunted growth and seed set failure, which may be caused by strong autotoxicity of heterologously produced phytochemicals against the tobacco itself. Panax ginseng roots contain various saponins (ginsenosides), which are major bioactive compounds. A monoglucosylated saponin, compound K (20-O-(ß-D-glucopyranosyl)-20(S)-protopanaxadiol), has high medicinal and cosmetic values but is present in undetectable amounts in naturally grown ginseng roots. The production of compound K (CK) requires complicated deglycosylation of ginsenosides using physicochemical and/or enzymatic degradation. In this work, we report the production of CK in transgenic tobacco by co-overexpressing three genes (PgDDS, CYP716A47 and UGT71A28) isolated from P. ginseng. Introduction and expression of the transgenes in tobacco lines were confirmed by genomic PCR and RT-PCR. All the lines of transgenic tobacco produced CK including its precursors, protopanaxadiol and dammarenediol-II (DD). The concentrations of CK in the leaves ranged from 1.55 to 2.64 µg/g dry weight, depending on the transgenic line. Interestingly, production of CK in tobacco brought stunted plant growth and gave rise to seed set failure. This seed set failure was caused by both long-styled flowers and abnormal pollen development in transgenic tobacco. Both CK and DD treatments highly suppressed in vitro germination and tube growth in wild-type pollens. Based on these results, metabolic engineering for CK production in transgenic tobacco was successfully achieved, but the production of CK and its precursors in tobacco severely affects vegetative and reproductive growth due to the cytotoxicity of phytochemicals that are heterologously produced in transgenic tobacco.

Exploring genes involved in benzoic acid biosynthesis in the Populus davidiana transcriptome and their transcriptional activity upon methyl jasmonate treatment.

Benzoic acids (BAs) are important structural elements in a wide variety of essential compounds and natural products, and play crucial roles in plant fitness. BA is a precursor of diverse benzenoid compounds, including the hormone salicylic acid (SA) and the aglycone moiety of salicin, which is particularly important in the Salicaceae family. The biosynthetic pathways leading to BA formation in plants are largely unknown. Recently, the CoA-dependent ß-oxidative BA biosynthesis pathway, which occurs in peroxisomes, has been characterized in petunia. The core of this pathway is cinnamic acid → cinnamoyl-CoA â†’ 3-hydroxy-3-phenylpropanoyl-CoA â†’ 3-oxo-3-phenylpropanoyl-CoA â†’ benzoyl-CoA. Here, we used 454 pyrosequencing to analyze the transcriptome of Populus davidiana and isolate putative genes involved in BA biosynthesis. De novo assembly generated 57,322 unique sequences, including 15,217 contigs and 42,105 singletons. From the unique sequences, we selected six genes exhibiting high similarity to genes encoding L-phenylalanine ammonia lyase, cinnamate:CoA ligase, cinnamoyl-CoA hydratase-dehydrogenase, 3-ketoacyl-CoA thiolase, benzoyl-CoA:benzyl alcohol O-benzoyltransferase, and benzaldehyde dehydrogenase. Each of these enzymes might be involved in BA biosynthesis. Real-time PCR (qPCR) analysis revealed that these six genes were highly transcribed in the aerial organs of P. davidiana, particularly in leaves. Treating the leaves of in vitro cultured plants with methyl jasmonate (MeJA) strongly enhanced the mRNA accumulation of all 6 genes, and this treatment also clearly enhanced the accumulation of BA, SA, salicyl alcohol, benzyl alcohol, benzyl benzoate, and benzaldehyde but not salicin. Our study shows that P. davidiana may possess a CoA-dependent ß-oxidative BA synthesis pathway. We also identified a relationship between the transcription of these genes and the accumulation of benzenoids, including BA and SA, which are highly responsive to the defense signaling molecule (MeJA).

Transcriptomic analysis of Siberian ginseng (Eleutherococcus senticosus) to discover genes involved in saponin biosynthesis.

BACKGROUND: Eleutherococcus senticosus, Siberian ginseng, is a highly valued woody medicinal plant belonging to the family Araliaceae. E. senticosus produces a rich variety of saponins such as oleanane-type, noroleanane-type, 29-hydroxyoleanan-type, and lupane-type saponins. Genomic or transcriptomic approaches have not been used to investigate the saponin biosynthetic pathway in this plant. RESULT: In this study, de novo sequencing was performed to select candidate genes involved in the saponin biosynthetic pathway. A half-plate 454 pyrosequencing run produced 627,923 high-quality reads with an average sequence length of 422 bases. De novo assembly generated 72,811 unique sequences, including 15,217 contigs and 57,594 singletons. Approximately 48,300 (66.3%) unique sequences were annotated using BLAST similarity searches. All of the mevalonate pathway genes for saponin biosynthesis starting from acetyl-CoA were isolated. Moreover, 206 reads of cytochrome P450 (CYP) and 145 reads of uridine diphosphate glycosyltransferase (UGT) sequences were isolated. Based on methyl jasmonate (MeJA) treatment and real-time PCR (qPCR) analysis, 3 CYPs and 3 UGTs were finally selected as candidate genes involved in the saponin biosynthetic pathway. CONCLUSIONS: The identified sequences associated with saponin biosynthesis will facilitate the study of the functional genomics of saponin biosynthesis and genetic engineering of E. senticosus.

Production of the dammarene sapogenin (protopanaxadiol) in transgenic tobacco plants and cultured cells by heterologous expression of PgDDS and CYP716A47.

KEY MESSAGE: Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.

Dammarenediol-II Prevents VEGF-Mediated Microvascular Permeability in Diabetic Mice.

Diabetic retinopathy is a major diabetic complication predominantly caused by vascular endothelial growth factor (VEGF)-induced vascular permeability in the retina; however, treatments targeting glycemic control have not been successful. Here, we investigated the protective effect of dammarenediol-II, a precursor of triterpenoid saponin biosynthesis, on VEGF-induced vascular leakage using human umbilical vein endothelial cells (HUVECs) and diabetic mice. We overproduced the compound in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase gene and purified using column chromatography. Analysis of the purified compound using a gas chromatography-mass spectrometry system revealed identical retention time and fragmentation pattern to those of authentic standard dammarenediol-II. Dammarenediol-II inhibited VEGF-induced intracellular reactive oxygen species generation, but it had no effect on the levels of intracellular Ca(2+) in HUVECs. We also found that dammarenediol-II inhibited VEGF-induced stress fiber formation and vascular endothelial-cadherin disruption, both of which play critical roles in modulating endothelial permeability. Notably, microvascular leakage in the retina of diabetic mice was successfully inhibited by intravitreal dammarenediol-II injection. Our results suggest that the natural drug dammarenediol-II may have the ability to prevent diabetic microvascular complications, including diabetic retinopathy.

Production of dammarenediol-II triterpene in a cell suspension culture of transgenic tobacco.

Dammarenediol-II is biologically active tetracyclic triterpenoid, which is basic compound of ginsenoside saponin. Here, we established the dammarenediol-II production via a cell suspension culture of transgenic tobacco overexpressing PgDDS. Dammarenediol-II synthase catalyzes the cyclization of 2,3-oxidosqualene to dammarenediol-II, which is the basic triterpene skeleton in dammarene-type saponin (ginsenosides) in Panax ginseng. Dammarenediol-II is a useful candidate both for pharmacologically active triterpenes and as a defense compound in plants. Dammarenediol-II is present in the roots of P. ginseng in trace amounts because it is an intermediate product in triterpene biosynthesis. In this work, we established the production of dammarenediol-II via cell suspension culture of transgenic tobacco. The dammarenediol-II synthase gene (PgDDS) isolated from P. ginseng was introduced into the Nicotiana tobacum genome under the control of 35S promoter by Agrobacterium-mediated transformation. Accumulation of dammarenediol-II in transgenic tobacco plants occurred in an organ-specific manner (roots > stems > leaves > flower buds), and transgenic line 14 (T14) exhibited a high amount (157.8 µg g⁻¹ DW) of dammarenediol-II in the roots. Dammarenediol-II production in transgenic tobacco plants resulted in reduced phytosterol (ß-sitosterol, campesterol, and stigmasterol) contents. A cell suspension culture was established as a shake flask culture of a callus derived from root segments of transgenic (T14) plants. The amount of dammarenediol-II production in the cell suspension reached 573 µg g⁻¹ dry weight after 3 weeks of culture, which is equivalent to a culture volume of 5.2 mg dammarenediol-II per liter. Conclusively, the production of dammarenediol-II in a cell suspension culture of transgenic tobacco can be applied to the large-scale production of this compound and utilized as a source of pharmacologically active medicinal materials.

A Pinus strobus transcription factor PsbHLH1 activates the production of pinosylvin stilbenoids in transgenic Pinus koraiensis calli and tobacco leaves.

Transcription factors (TFs) play an important role in regulating the biosynthesis of secondary metabolites. In Pinus strobus, the level of methylated derivatives of pinosylvin is significantly increased upon pine wood nematode (PWN) infection, and these compounds are highly toxic to PWNs. In a previous study, we found that the expression of a basic helix-loop-helix TF gene, PsbHLH1, strongly increased in P. strobus plants after infection with PWNs. In this study, we elucidated the regulatory role of the PsbHLH1 gene in the production of methylated derivatives of pinosylvin such as pinosylvin monomethyl ether (PME) and dihydropinoylvin monomethyl ether (DPME). When PsbHLH1 was overexpressed in Pinus koraiensis calli, the production of PME and DPME was significantly increased. Overexpression of the stilbene synthase (PsSTS) and pinosylvin methyl transferase (PsPMT) genes, known as key enzymes for the biosynthesis of methylated pinosylvins, did not change PME or DPME production. Moreover, PME and DPME were not produced in tobacco leaves when the PsSTS and PsPMT genes were transiently coexpressed. However, the transient expression of three genes, PsSTS, PsPMT, and PsbHLH1, resulted in the production of PME and DPME in tobacco leaves. These results prove that PsbHLH1 is an important TF for the pinosylvin stilbene biosynthesis in pine plants and plays a regulatory role in the engineered production of PME and DPME in tobacco plants.

Tobacco NtLTP1, a glandular-specific lipid transfer protein, is required for lipid secretion from glandular trichomes.

Glandular trichomes are the phytochemical factories of plants, and they secrete a wide range of commercially important natural products such as lipids, terpenes and flavonoids. Herein, we report that the Nicotiana tabacum LTP1 (NtLTP1) gene, which is specifically expressed in long glandular trichomes, plays a role in lipid secretion from trichome heads. NtLTP1 mRNA is abundantly transcribed in trichomes, but NtLTP3, NtLTP4 and NtLTP5 are not. In situ hybridization revealed that NtLTP1 mRNAs accumulate specifically in long trichomes and not in short trichomes or epidermal cells. X-gluc staining of leaves from a transgenic plant expressing the NtLTP1 promoter fused to a GUS gene revealed that NtLTP1 protein accumulated preferentially on the tops of long glandular trichomes. GFP fluorescence from transgenic tobacco plants expressing an NtLTP1-GFP fusion protein was localized at the periphery of cells and in the excreted liquid droplets from the glandular trichome heads. In vitro assays using a fluorescent 2-p-toluidinonaphthalene-6-sulfonate probe indicated that recombinant NtLTP1 had lipid-binding activity. The overexpression of NtLTP1 in transgenic tobacco plants resulted in the increased secretion of trichome exudates, including epicuticular wax. In transgenic NtLTP1-RNAi lines, liquid secretion from trichomes was strongly reduced, but epicuticular wax secretion was not altered. Moreover, transgenic tobacco plants overexpressing NtLTP1 showed increased protection against aphids. Taken together, these data suggest that NtLTP1 is abundantly expressed in long glandular trichomes, and may play a role in lipid secretion from long glandular trichomes.

The involvement of ß-amyrin 28-oxidase (CYP716A52v2) in oleanane-type ginsenoside biosynthesis in Panax ginseng.

Panax species are the most popular medicinal herbs. The root of these plants contains pharmacologically active triterpene saponins, also known as ginsenosides, compounds that are divided into dammarane- and oleanane-type triterpenes. Two CYP716A subfamily genes (CYP716A47 and CYP716A53v2) were recently characterized, encoding an enzyme catalyzing the hydroxylation of dammarane-type triterpenes in Panax ginseng. Herein, we report that one CYP716A subfamily gene (CYP716A52v2) isolated from P. ginseng encodes a ß-amyrin 28-oxidase, which is suggested to modify ß-amyrin into oleanolic acid, a precursor of an oleanane-type saponin (mainly ginsenoside Ro) in P. ginseng. The ectopic expression of both PNY1 and CYP716A52v2 in recombinant yeast resulted in erythrodiol and oleanolic acid production, respectively. In vitro enzymatic activity assays biochemically confirmed that CYP716A52v2 catalyzed the oxidation of ß-amyrin to produce oleanolic acid, and the chemical structure of the oleanolic acid product was confirmed using gas chromatography-mass spectrometry (GC/MS). Transgenic P. ginseng plants were generated via Agrobacterium tumefaciens-mediated transformation: the overexpression of CYP716A52v2 greatly increased the content of oleanane-type ginsenoside (ginsenoside Ro), whereas RNA interference against CYP716A52v2 markedly reduced it. Furthermore, the levels of other dammarene-type ginsenosides were not affected in these transgenic lines. These results indicate that CYP716A52v2 is a ß-amyrin 28-oxidase that plays a key role in the biosynthesis of oleanane-type triterpenes in P. ginseng.

Metabolite Profiling to Evaluate Metabolic Changes in Genetically Modified Protopanaxadiol-Enriched Rice.

Event DS rice producing protopanaxadiol (PPD) has been previously developed by inserting Panax ginseng dammarenediol-II synthase gene (PgDDS) and PPD synthase gene (CYP716A47). We performed a gas chromatography-mass spectrometry (GC-MS)-based metabolomics of the DS rice to identify metabolic alterations as the effects of genetic engineering by measuring the contents of 65 metabolites in seeds and 63 metabolites in leaves. Multivariate analysis and one-way analysis of variance between DS and non-genetically modified (GM) rice showed that DS rice accumulated fewer tocotrienols, tocopherols, and phytosterols than non-GM rice. These results may be due to competition for the same precursors because PPDs in DS rice are synthesized from the same precursors as those of phytosterols. In addition, multivariate analysis of metabolic data from rice leaves revealed that composition differed by growth stage rather than genetic modifications. Our results demonstrate the potential of metabolomics for identifying metabolic alterations in response to genetic modifications.

Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng.

Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs, and the root of this plant contains pharmacologically active components, called ginsenosides. Ginsenosides, a class of tetracyclic triterpene saponins, are synthesized from dammarenediol-II after hydroxylation by cytochrome P450 (CYP) and then glycosylation by a glycosyltransferase. Protopanaxadiol synthase, which is a CYP enzyme (CYP716A47) that catalyzes the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol, was recently characterized. Here, we isolated two additional CYP716A subfamily genes (CYP716A52v2 and CYP716A53v2) and determined that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in P. ginseng. Both CYP716A47 and CYP716A53v2 mRNAs accumulated ubiquitously in all organs of ginseng plants. In contrast, CYP716A52v2 mRNA accumulated only in the rhizome. Methyl jasmonate (MeJA) treatment resulted in the obvious accumulation of CYP716A47 mRNA in adventitious roots. However, neither CYP716A52v2 nor CYP716A53v2 mRNA was affected by MeJA treatment during the entire culture period. The ectopic expression of CYP716A53v2 in recombinant WAT21 yeast resulted in protopanaxatriol production after protopanaxadiol was added to the culture medium. In vitro enzymatic activity assays revealed that CYP716A53v2 catalyzed the oxidation of protopanaxadiol to produce protopanaxatriol. The chemical structures of the protopanaxatriol products were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Our results indicate that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that produces protopanaxatriol from protopanaxadiol, which is an important step in the formation of dammarane-type triterpene aglycones in ginseng saponin biosynthesis.

Dammarenediol-II production confers TMV tolerance in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase.

Panax ginseng is one of the famous medicinal plants. Ginsenosides, a class of tetracyclic triterpene saponins, are mainly responsible for its pharmacological activity. Most ginsenosides are composed of dammarenediol-II aglycone with various sugar moieties. Dammarenediol-II synthase is the first enzyme in the biosynthesis of ginsenosides. Here, we report that transgenic tobacco expressing the P. ginseng dammarenediol-II synthase gene (PgDDS) produced dammarenediol-II, and conferred resistance to Tobacco mosaic virus (TMV). Upon infection with TMV, lesions developed more rapidly in transgenic tobacco plants, and their size was smaller than those of wild-type plants. Transgenic tobacco plants showed a low level of both the viral titer and mRNA accumulation of TMV coat protein (CP) compared with the wild type. The production of dammarenediol-II in transgenic tobacco stimulated the expression of tobacco pathogenesis-related genes (PR1 and PR2) under both virus-untreated and -treated conditions. When the leaves of wild-type plants were inoculated with a mixture of TMV and dammarenediol-II, the leaves exhibited a reduced viral concentration and TMV-CP expression than those receiving TMV treatment alone. When the leaves of P. ginseng were infected with TMV, transcription of PgDDS was significantly increased. Transgenic P. ginseng plants harboring a ß-glucuronidase (GUS) gene driven by the PgDDS promoter were constructed. The GUS expression was activated when the transgenic ginseng plants were treated with TMV. These results indicate that the medicinally important dammarenediol-II can be ectopically produced in tobacco, and the production of dammarenediol-II in tobacco plants allows them to adopt a viral defense system.

Production of Nematicidal Pinosylvin Stilbenes in Cell Suspension Cultures of Pinus koraiensis by Fungal Elicitation.

Pinosylvin stilbenes are natural phenolic compounds found in the Pinaceae family and act as phytoalexins. Some pinosylvin stilbenes have strong nematicidal activity against pine wood nematodes (PWNs: Bursaphelenchus xylophilus). Here, we established the efficient production of two pinosylvin stilbenes, dihydropinosylvin monomethylether (DPME) and pinosylvin monomethylether (PME), by cell suspension culture of Pinus koraiensis after fungal elicitation. DPME and PME were found in small amounts (less than 40 µg/g DW) in the stem bark and leaves of P. koraiensis plants. Cell suspension cultures were established from the cultures of calli derived from mature zygotic embryos of P. koraiensis in 1/2 Litvay medium containing 2.2 µM 2,4-D and 2.2 µM BA. Two types of fungal elicitors, fungal cell extract (CE) and fungal medium filtrate (MF), were prepared from three species of fungi (Penicillium chrysogenum, P. pinophilum, and P. roquefortii). CE and MF treatments strongly stimulated the production of PME and DPME in cultured cells. The production of PME in suspension cells of P. chrysogenum, P. pinophilum, and P. roquefortii MF treatments after 3 days was 5734 µg/g DW, 4051 µg/g DW, and 6724 µg/g DW, respectively. Pinosylvin synthase (PkSTS) and pinosylvin O-methyltransferase (PkPMT) are key genes in DPME and PME biosynthesis. qPCR analysis revealed that the expression of the PkSTS and PkPMT in cultured cells was highly enhanced after fungal elicitor treatment. The cell extracts after MF treatment resulted in 92.5 ± 7.8% immobilization of the adult PWNs and 63.7 ± 3.5% immobilization of the juvenile PWNs within 24 h. However, control cell extracts without MF treatment showed 11.3 ± 1.4% nematicidal activity against adult PWNs. Our results suggest that pinosylvin stilbenes can be produced from the cell culture of P. koraiensis after fungal elicitor treatment and can be used as nematicidal compounds against PWNs.